And IL-7Tg Mice. Spleen Cells Were Pr

SUPPLEMENTAL INFORMATION

10.0 Adult wildtype 8.0 Young wildtype

6.0

4.0

sement usage 2.0 H 1.0

% V 0.5

0.0 1-61

VH8-8 VH8-6 VH8-5 VH8-4 VH1-9 VH1-7 VH1-5 VH1-4 VH6-7 VH6-6 VH6-5 VH6-4 VH6-3 VH3-8 VH9-4 VH3-6 VH3-5 VH3-4 VH7-4 VH3-3 VH7-3 VH9-3 VH9-2 VH9-1 VH3-2 VH4-2 VH3-1 VH4-1 VH7-2 VH7-1 VH2-9 VH2-7 VH2-6 VH2-5 VH5-9 VH2-4 VH5-6 VH2-3 VH5-4 VH2-2 VH5-2 VH1-85 VH1-84 VH1-82 VH1-81 VH1-80 VH1-78 VH1-77 VH1-76 VH1-75 VH1-74 VH1-72 VH1-71 VH8-12 VH1-69 VH1-67 VH1-66 VH8-11 VH1-64 VH1-63 VH1-59 VH1-58 VH1-56 VH1-55 VH1-54 VH1-53 VH1-52 VH1-50 VH1-49 VH1-47 VH1-43 VH1-42 VH1-39 VH1-37 VH1-36 VH1-34 VH1-31 VH1-26 VH1-22 VH1-20 VH1-19 VH1-18 VH1-15 VH1-14 VH1-12 VH1-11 VH15-2 VH10-3 VH10-1 VH12-3 VH13-2 VH13-1 VH14-4 VH16-1 VH14-3 VH11-2 VH14-2 VH11-1 VH14-1 VH5-17 VH5-16 VH5-15 VH5-12 VH2-6-8 VH2-9-1 VH5-9-1 VH VH1-62-3 VH1-62-2 VH1-62-1 VH1-17-1 VH5-12-4

10.0 Adult wildtype -/- 8.0 Adult IL-7

6.0

4.0

sement usage 2.0 H 1.0

% V 0.5

0.0 1-61

10.0 Young wildtype Young IL-7Tg 8.0

6.0

4.0

sement usage 2.0 H 1.0

% V 0.5

0.0 1-61

-/- Supplemental Figure 1. VH gene segment usage inDickinson wildtype, et al., Suppl.IL-7 Fig.1 and IL-7Tg mice. Spleen cells were prepared from adult wildtype (n=4); young wildtype (n=7); adult IL-7-/- (n=7) and young IL-7Tg (n=5) mice and stained with a cocktail of fluorescent antibodies specific for surface CD19, B220, and IgM. One to two million B cells (CD19+ B220+ IgM+/-) were FACS sorted and subjected to mouse IgH gene expression analysis as described in materials and methods. We used C57BL/6 VH gene nomenclature from the International ImMunoGeneTics ® ® information system (IMGT ; http://www.imgt.org). All of the 113 functional VH gene segments in C57BL/6J mice were arranged from 5’ to 3’ in the IgH locus (i.e., distal to proximal orientation with respect to the DH-JH gene region of the IgH locus). The frequency of VH segment usage was calculated as percent from one million IgH sequences.

Supplemental Figure 2. Characterization of phenol-chloroform extracted B1355 dextran. To eliminate TLR ligand contamination, whole peritoneal cavity cells of naïve C57BL/6J mice were incubated either with various concentrations of crude bacterial B1355 preparation or with B1355 obtained from three rounds of Phenol-extraction as previously described (37). As a positive control, LPS (100ng/ml, TLR4 agonist) Dickinson was used. et.After al., 18 Suppl-24 hours, Fig. t he2 culture supernatants were subjected to IL-6 ELISA as a readout to measure the degree of TLR ligand contamination. The data is representative of two independent experiments.

A. C57BL/6J 100 T 34±4100 B1a 110±17100 B1b 77±12100 B2 71±9

80 1704±57 80 94±9 80 65±8 80 62±6

60 60 60 60

40 40 40 40 % of Max %of 20 20 20 20

0 0 0 0 2 3 4 5 0 102 103 104 105 0 10 10 10 10 0 102 103 104 105 0 102 103 104 105 B. BALB/cJ 100 T 54±4100 B1a 75±22100 B1b 42±22100 B2 43±17 80 1124±229 80 75±19 80 41±21 80 41±16

60 60 60 60

40 40 40 40

% of Max %of 20 20 20 20

0 0 0 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 IL-7Rα

Supplemental Figure 3. Mature B cells do not express IL-7Rα. Peritoneal cavity cells of adult naïve C57BL/6J (n=5) or BALB/cJ (n=5) mice were stained with antibodies specific for surface IgM, CD19, Mac1, CD5, and IL7Rα, and analyzed by flow cytometry to assess IL7Rα surface expression onDickinson T cells (CD19 et. al.,-, IgM Suppl.-, CD5 Fig.high ),3 B1a (CD19+, IgM+, CD5+, Mac1+), B1b (CD19+, IgM+,CD5-, Mac1+), and B2 (CD19+, IgM+, CD5-, Mac1-) subsets in the peritoneal cavity(plots not shown). Histograms depicting IL-7Rα expression (black line) in each cell subset compared to isotype control (gray lines) are provided. Mean fluorescent intensity ± standard deviation of IL-7Rα-specific antibody binding is provided in each plot (black number) as compared to unstained control (gray number).

Supplemental Figure 4. α1,3-dextran-specific polysaccharide-specific B cell expansion does not require IL-7. Expansion of α1,3-dextran-specific (EB3-7+Dickinson) peritoneal B et. cell al., subsets Suppl in MK7 Fig.-immunized 4 -/- VHJ558 Tg x IL-7 mice was measured by flow cytometry as described in the Figure 7 legend and absolute cell counts were calculated. Statistical significance was determined using Mann- Whitney test with p values shown in the plot.